1. Field of Invention
The invention deals with intracellular protein delivery vehicles, methods of preparing them, and their use to deliver proteins to a cellular target.
2. Discussion of Related Art
An average eukaryotic cell contains thousands of different proteins that participate in normal cellular functions. Most human diseases are somehow related to malfunctioning of particular proteins systematically or locally. In this context, protein therapy (Birch et al., Therapeutic Proteins, methods and Protocols, (Humana Press, Totowa), pp. 1-16 2005) offers the most direct and safe approach for the treatment of such diseases. Recent advances in recombinant DNA technology enables the synthesis of a large variety of pharmaceutical proteins (Nagle et al., Nature Rev. Drug Discov., vol. 2, pp. 75-79, 2003; Brekke et al., Nature Rev. Drug Discov., vol. 2, pp. 52-62, 2003); however, broad use of the protein therapy is still limited by several substantial technical barriers, such as low efficiency of intracellular delivery and poor stability of protein against proteases.
Intracellular use of therapeutic proteins is of great importance for treatments of cancers and protein-deficient diseases (Wadia et al., Protein Transport (Cell-penetrating peptides: Processes and Applications, (CRC Press), p. 365, 2002). However, compared with the wide applications of extracellular active proteins, intracellular active protein drugs are rare in clinical applications (Birch et al., Therapeutic Proteins, methods and Protocols, (Humana Press, Totowa), pp. 1-16 2005), partially due to their poor stability in serum and weak permeability through cell membrane. Although some proteins can be translocated from the extracellular space into cells by receptor-mediated endocytosis (Vyas et al., Crit. Rev. Ther. Drug Carrier Syst., vol. 18, pp. 1-76, 2001; Sato et al., Adv. Drug Deliv. Rev., vol. 19, pp. 445-467, 1996), they may be entrapped within the endosome and degraded in the lysosome rather than be released to the appropriate cellular compartment. There is a growing effort to circumvent this problem. For example, liposome-wrapped proteins were shown to be transferred into the cytoplasm but with a low efficiency (Straubinger et al., FEBS Lett., vol. 179, pp. 148-154, 1985; Bulmus et al., J. Control Release, vol. 93, pp. 105-120, 2003). Recently, several small cationic peptides (termed cell-penetrating peptides, CPPs) were identified and used to assist protein delivery (Fawell et al., Proc. Natl. Acad. Sci. USA, vol. 91, pp. 664-668, 1994; Morris et al., vol. 19, pp. 1173-1176, 2001); Schwarze et al., Science, vol. 285, pp. 1569-1572, 1999). With significantly improved intracellular delivery efficiency and retained activity in cells, this strategy is promising for pharmaceutical application. In addition, although one can deliver proteins into cells with these strategies, the poor stability of proteins within the cells still hampers the wide applications of therapeutic proteins. Among various possible inactivation factors (Fagain et al., Biochim. Biophys. Acta., vol. 1252, pp. 1-14, 1995), protease digestion in serum is a crucial one, which needs to be addressed to ensure a successful intracellular protein delivery (Hooper, N. M. Proteases in Biology and Medicine, (Portland Press, London), 2002).